The objectives of this project are to study the cartilage and bone inducing factors and to define their role in embryogenesis and in postnatal life, both in tissue formation and in disease. As tissue regeneration recapitulates the developmental sequence of embryonic tissue formation, it is conceivable that understanding the mechanisms of action of the soluble differentiation factors is a key step towards biologically controlled regeneration of skeletal tissues. This will have a significant impact on the treatment of congenital and/or acquired skeletal diseases such as large bone defects, impaired fracture healing, osteoarthritis, osteoporosis and periodontitis. This project focuses on the further characterization of cartilage and bone inducing molecules and their biological activities. Protein fractions with cartilage and bone inducing activity in vivo, have been isolated from articular cartilage and purified to homogeneity; protein sequencing data from tryptic peptides have been obtained; databank searches did not reveal homology with any known sequences. The tissue specific localization of the new protein preparation was shown by immunolocalization using a polyclonal antibody against the N-terminal sequence. Further characterization by cDNA cloning is in progress. Recombinant transient expression of isolated cDNA clones is ongoing. Using the molecular probes, we are studying their respective contribution to cartilage, endochondral and membranous bone formation. Immunohistochemical localization and in situ hybridization of cartilage and bone inducing proteins, as well as studies in vitro, indicate the selective contribution of these molecules to initiation, enhancement, maintenance and maturation of the chondrocytic and osteoblastic phenotype. This work contributes to the basic understanding of de novo cartilage and bone formation, and sets the stage for the proper indication and use of these soluble differentiation factors in a clinical setting.